 So, label-free biosensors have revolutionized our ability to look at protein-protein and protein-other biomolecular interactions in label-free manner essentially in the real time with very, very high throughput. There are different technologies which are started showing its promises. We have discussed about SPR or surface plasma resonance. Another promising technology is BLI or Biolayer Interferometry. The kinetic analysis of antibodies and other proteins is critical to the characterization of molecules. In today's lecture, Mr. Sushil Vedya, an application scientist of Paul Bioforte, will use Biolayer Interferometry or BLI technique to perform the kinetic interaction between the mouse monoclonal antibody and protein A. Here the protein A will be immobilized on a matrix at the tip of a fibre optic sensor. The binding between the immobilized ligand and the analyte, for example, the mouse monoclonal antibody will produce a change in the optical thickness at the tip and relating in the wavelength shift which will be proportional to the binding. The data acquisition and data analysis will also be demonstrated by determination of interaction kinetics between the ligand and the analyte. So, let us start this demonstration session and today's lecture. Myself Sushil Indra Vedya, application specialist for the Forte Bio Paul Corporation. Just now in the last talk, Dr. Shingalan Kaur spoke about the Biolayer Interferometry and the applications. Now I am going to demonstrate how this Biolayer Interferometry works. Now I am going to demonstrate the kinetic studies of the monoclonal antibodies that is the mouse monoclonal antibody to the protein A ligand. This is the Paul Forte Bio Octate Red 96 instruments. If you look at inside, this is we call it as optical head box. The optical head box consists of a spectrophotometer as well as the channels or we call it as a manifold. This is 8 channel manifold, so it can take up the 8 sensors. So here if you look at this is the sensor compartment, this is we call it as a sample compartment. Now you can see this, this is we call it as a sensor holder or sensor manifold. So here we can put in this sensor in this fashion and then we can put it this as a sample. So this is the 96 well plate format, we can analyze 96 samples in one goal. If you look at this, this is works on the principle of the depend rate as well as the Biolayer Interferometry. You know about the principle behind the Biolayer Interferometry in the last talk. Here it consists of a white light as if you look at here this is called as the white light. You can able to see here, this is the light emitting diode, we are passing the light emitting diode, the light pass through the sensor. If you look at this, this is the sensor, it is actually made up of the glass, it consists of a fibre optic inside and at the holder part this is made up of the plastic. So inside there is a fibre optic, at the tip of the sensor we are putting ligand of our interest. So we are passing a white light, light get reflected back. It is a reflection based phenomenon. One light which is coming from the internal optical layer, another light which is coming from the ligand end. So when there is an interaction between the two binding partners, say suppose I am coating with the kind of a matrix, it is a protein A. When you dip into the sensor into the corresponding binding partner example like monoclonal antibody, when human IgG starts binding to the protein A matrix, as you know that it is a concentration dependent as the more molecule starts binding to the protein A matrix, then we can able to see there is a change in the spectrum or I mean to say it is like a change in the wavelength. So the change in the wavelength is nothing but the, as the concentration increases the change in the wavelength, it is corresponds to the optical thickness. The more the optical thickness as we can see as the change in the wavelength, it indicates that the more the molecules are binding. So from this we can able to like determine the concentration. This instrument even we can useful for the quantification purpose, say suppose in case of the industry or any protein if you want to do a quantification directly we can take a known concentration of the standards, we can generate a standard curve and from we can able to determine the unknown concentrations. Apart from that the major useful of this instrument is to study the binding kinetics where we will determine the rate constants like on rate, off rate as well as the affinity constants. I am going to demonstrate you kinetics introduction of the most monoclonal antibody to the protein A ligand. Here I am using biotinylated protein A as a ligand at the concentration I am using a 10 microgram per ml, 10 microgram per ml I am putting into the 6 wells and most monoclonal antibody I am using as a analyte. Here I am taking the starting concentration at 25 microgram per ml I am going to do a serial delusion with a 2 fold from 25 to 12.5 then serially I go for a 2 fold concentration around 6 data points. This is the plate map here, this is the buffer the first column and the second column I am going to put the 10 microgram per ml concentration of a biotinylated protein A and the third column I am going to buffer it will be useful as a for an unknown biotinylated protein A get washed off here and also this well I am going to useful for the dissociation purpose and the last well I am going to put the analyte that is the 25 microgram per ml serially deluted in a all these 6 wells. This is the sample plate 96 well plate I am going to place in the sample compartment here like this there is a click sound it will come firmly it fits into the sample compartment here the sample compartment if you look at its works on the depend read the BLI technology works on the depend read method here we do not have a like a any microfluidic devices so to assist for a binding we have a like a shaker that shaker assess in the binding the shakers works from 150 rpm to 1500 rpm and also this sample plate have a like there is a temperature we can work from the ambient room temperature like 20 23 plus 4 degree to the 40 degree centigrade. So here I am going to place the sensor compartment so we can from we pick up the sensors from the either column one column two or column three like anywhere we can make it program according to the in the instrument. We have placing the sensor rack like this if you look at this in the in the software there is a we have a in a 45 platform the BLR technology we have a acquisition software as well as the data analysis software now I will show you the acquisition software what are all the features it have if I double click on this instrument starts initializing if you look at its moves in the XYZ direction itself it initializes okay now we can see that there is a initialization process is going on it takes a roughly around 30 seconds of time. So you can see in the software what the event is happening on the dialogue box open now it is showing that the instrumentate status it is ready so now we can start the experiments when there is a acquisition software open we can see there is a experimental wizard here so here in the wizard we can see there is a two major experiments whether you want to do a kinetics experiments or the quantitation experiments now we are going to perform the kinetics experiments I will go here clicking on the screen are a mark so if you look at this there is a page get opened here if you see on your left hand side there is a 96 well plate design this is what we call it as a plate design we had to mention in the plate design what things we had put it so I had mentioned we had put a like a leaguer biotinulated ligand buffers as well as the the twofold dilutions of the different twofold dilution concentration of the most mocha antibody now we have to mention where all these things we had put in the 96 well plate so I had put a in the 96 well plate here this column I put it as a buffer I will right click it here right click I am showing this as a buffer here second column I had put a biotinulated ligand that we call it as a load load is nothing but we are performing the biotinulated protein a immobilization step we are performing on the stripped iodine sensor now the third column I had put here this one as a buffer once again unbound material get washed off here now we have to put analyte we call it as a sample so these are the sample ideas we have to mention what we had put it here we can put it this here just like a buffer so what I will do is just I will take I will copy it then buffer and once again here I had put a once again there is a buffer here I put a protein a as a ligand here as I mentioned the buffer we can use any kind of buffer now in this experiment I am using a PBS buffer having a additive like a BSA it is a 0.1% BSA as well as it having a like a surfactant like a 0.02% of the between 20 so this this composition P in a PBS at the pH 7.2 so I am here I am mentioning the mouse monochrome antibody I will use M small g capital G MIGG okay I am using a 25 microgram per ml and the molecular weight of the mouse molecule antibodies 150 kilo Dalton it is automatically calculate it is a 166.7 nano molar okay just I will copy this one what I will do I will I will select this such well data then it is a 25 divided by 2 then automatically last one I am put it as a 0 concentration these are the concentrations 25 12.5 6.25 3.1 2 5 1.56 and 0.7 corresponding nano molar concentration now we had in the there is one more step we call it as a assay definition so these are the some of the steps I am going to add here there is a baseline by default baseline just acquires the buffer now we are going next step we call it as a loading loading is nothing but we are immobilizing a 10 microgram per ml protein a on to the iodine sensor so I am going to add here then next step I am performing the baseline and I will say it as a okay then instrument says that already there is a one more first already we have a baseline there is a exist it is saying do you need a one more yes I need one more I will say okay then I will add one more step we call it as association steps because okay then second we call it as a dissociation step okay now we have to tell instrument what is the first step this is the first step baseline I am going here I am double click on that this is the first step we call it as a baseline step the second step we call it as immobilization protein a immobilized biotinylated protein a immobilization on to the stripped iodine sensor I will double click on this then the third step baseline once again unbound material get washed off here just I am clicking on this next step is the association step this is the sample this is the mouse mocha antibodies to fold diluted six data points then the dissociation step I am performing a same buffer that is the column 4 okay these are the states what sense what type of sensors we are using I am using the stripped iodine sensor now we have to say for an instrument how much time each step has to be acquired the first step I require around 30 seconds and the second step it is require around once again the 30 seconds third step it is a good to have 60 seconds association I will perform for a 150 seconds and the dissociation I will perform for around 200 seconds this is for this experiments these are the conditions we already pre-optimized so for this it is well established I am performing and here I am performing the shake speed at the 1000 rpm if you look at the total time it is roughly around 9 minutes and now we have to say in the in the sensor compartment from where the sensor has to be pick up now I had put a sensor somewhere here at the third column in the sensor compartment if you look at the next step review experiment in the review it is like that your sensor is picking from the third column then the first step will be your baseline second step here was loading third step your baseline and the fourth step your association then the fifth step your baseline so run experiment then where exactly want to store the data here I am going to store the data in the desktop have IIT demo I am creating this folder whenever you are performing the experiment sensors has to be hydrate prior to the experiment at least 10 minutes it is require for the hydrations say suppose your sensors are not hydrated we can say in the software delay 600 seconds 600 seconds is nothing but a 10 minutes after the hydration the sensors the instrument starts acquisition in second option is like that during the hydration whether your sample has to be in the shaking conditions because the instrument have a shaker it will during the effectively mix in some cases it is required some cases not you know that shaking sometimes enhances the aggregation so we can uncheck this and also I think prior to the experiment we already started a hydration of the sensors now sensors got hydrate we can directly uncheck this we can go for the experiment and sample plate temperature it is at the 30 degree so already it is stabilized the plate we can now go for the experiment here if you look at this I open the door for the demonstration purpose in all the cases we have to close this door because trail it affects the interactions now for the demonstration purpose I am keep opening the door I will say go sensor is speaking from here from the sensor rack it is now it is going to the sample plate sample plate if you look at now there is a shaker which is working at the 1000 rpm so you can you could able to see there is a as when it dips into the sample compartment you see there is a signal so now this signal we are acquiring for the 30 seconds the first step we called as a baseline now it is going to a next compact next column it is consists of a biotinylated protein at n microgram per ml now we can able to see there is a rise in the signal this is what we call it as a immobilization step the biotinylated protein is binding to the the striptavidin sensor now that is we are acquiring for around roughly a 30 seconds time and it is around in in less than 20 seconds we can able to see it is reaching to the 1 nanometer the binding we are measuring in terms of the nanometer here this is a good enough loading for to get the interaction studies now the moving to the next column where there is a buffer PBS buffer unbound material get washed off and in this steps is required for the to get a stabilized baseline the baseline is now stabilized we can go from here we can say okay it is automatically goes to the next step 30 seconds now the sensors are moving to the association step now here there is a biotinylated protein a interacting to your most macro antibody it is like a two-fold different concentrations if you look at this the graphs this is how the curves are two fold dilutions this is the highest concentrations this is the second fold this is third fold this is fourth one at the the bottom I had put the zero concentration so in a software we have a function like that we can extend the current step as well as if you want like it is already like a saturated we can go to the next step I will say go to the next step 100 seconds is good the sensors went back to the buffer well where exactly if there is a interactions happening if it is a loosely bound kind of interactions just get come out from the sensor if it is a very strong then you can able to see there is a still kind of a straight line here so if it is a straight line then it indicates that it is a very strong binding once the dissociation step performed the sensors once again rewrack back to the sensor tray and and the instrument the robotic arm moved to its original position so now acquisition done now we have to go for the data analysis there is a data analysis software I am going to double click on the data analysis software in the bottom of this where exactly you have a data so I it be demo I will I will go with this kinetics demo I have a folder here this I am double click on this if you see that there is a page got open I will go to processing here in the processing step you will see this the raw data so what we are doing is we are subtracting I had like a one of the column I had put a 0 concentration this column I will change change to the reference well this is what I am 0 concentration because some of the artifacts from the buffer we can subtract from the data so that is why I am going for the reference subtractions on the on the left hand side there is a here we have icon subtraction we have different types of the subtraction systems so I am using here one of the just only a 0 concentration I am using for the subtraction so reference well I am going for that in the raw data I will go that I will select this association step then there is a Y align Y axis I am going to do a baseline this is the baseline step for this particular baseline I am going to align now I have to go for the process data so if you look at now the data what we got this is what the process data now we have to go for the analysis analysis there is a the six data points we have here in the analysis part we have association only dissociation only association and the dissociation for to calculate the rate constraints we have to go always choose the association and the dissociation we have a different binding models like 1 is to 1 2 is to 1 for the heterogeneous ligand mass transport effect if there is any mass transport involving the interaction studies we have a bivalent analyte 1 is to 2 binding model depletion studies is so local in the fitting model apart from the what kind of model we have a like another kind of a fitting it is a local fitting as well as global fitting the most of the times when you are performing for the interaction studies we always go with the global fitting so now I have to choose a global fitting global fitting with respect to color and the r max unlinked by a sensor to perform a global fitting we have to select all the samples change to a one color so I will change to a blue color now you can see this now is a 1 is to 1 binding model I had to try use entire steps systems I have to fit the curve so now fitting is okay and the KD it is showing that around a 17 nano mole easily we can able to see the good fits through the with respect to the chi square values as well as the R square values if you look at in this table we have a like what is the KD the KD errors K on with the K on errors and if you look at the good fits it is always when in case of the kinetics the good fit comes it is a 0.5 and 0.95 above and also quiet chi square should be less than 3 so from this indicating that it is behaves a 1 is to 1 binding model it is a it is supported by a your R square as well as the chi square the statistical parameters also we have a graphs like XY graph iso affinity graphs as well as the steady state because in this experiments we not reach the steady states we not able to like a calculate the steady states but still software can pick up and the KD value it is determining it is around a 2.1 nano molar and also here also if you look at this is around roughly a 1.7 nano molar it is almost like a similar very near to those values yes it is a good fit and we can export this the into a printed versions like a making a report here we have to say save report then say export we can take like a kind of excel sheet what the parameters I had used for that experimental summary the sensor tray where exactly we had put a sensors the sensor data what sensor we had used sample data how your sample plate design is raw data you can see the raw data aligned data as well as the process data how this we had subtracted using a reference well subtraction method the graphs the fitted graphs the residual view versus the experimental view the blue ones which are the experimental red ones which are the theoretical in the results tables what is the where the sensor locations sample ID the concentrations response what we got what is the KD KD errors so all entire details we can get it into the report now we successfully demonstrated the protein-protein interactions here with the protein a with the most small quantity bodies we used and we got a very good data with the one is to one binding model with the affinity is around 1.7 nano molar range so with this I am concluding my demonstrations I hope you got a glimpse of how to perform these biomolecular interaction studies using bio layer interferometry platform today we have witnessed the application of BLI to measure the interaction between protein a and mouse monoclonal antibody as demonstrated the system monitored association of the analyte with the immobilized ligand and dissociation after moving the sensor to the solution without the binding partner the changes in interference pattern where quantified and use to determine the kinetic rate of binding and dissociation in the next lecture we will demonstrate another application of bio layer interferometry based label free application for the quantification of proteins thank you.